Use of lecithin-cholesterol acyltransferase (LCAT) to reduce accumulation of cholesterol

ABSTRACT

This invention provides methods for treating atherosclerosis in a mammalian subject by increasing the activity of LCAT in the serum of the subject to a level effective to decrease the accumulation of cholesterol in the subject. Pharmaceutical dosage forms containing LCAT also are provided.

This is a U.S. national phase of PCT/US96/18195, filed Nov. 8, 1996,which claims priority to U.S. provisional application No. 60/006,400,filed Nov. 9, 1995.

BACKGROUND OF THE INVENTION

This invention relates to methods for the prophylactic and therapeutictreatment of atherosclerosis and to diseases relating to a deficiency inlecithin-cholesterol acyltransferase activity.

Atherosclerosis is a pathological condition of mammals characterized bythe accumulation of cholesterol in the arteries. Cholesterol accumulatesin the foam cells of the arterial wall, thereby narrowing the lumen.This results in decreased flow of blood. The clinical sequelae ofatherosclerosis include heart disease and heart attack, stroke, andperipheral vascular disease. Together, these diseases account for moredisease-related deaths in industrialized countries than any other cause.

The development of human atherosclerosis is inversely related to theconcentration of high density lipoproteins (HDL) in the serum. D. J.Gordon and B. M. Rifkind (1989) N. Engi. J. Med. 321:1311. Highconcentrations of HDL appear to protect against the development ofpremature atherosclerosis, while low HDL cholesterol concentrations areassociated with an increased risk of cardiovascular disease. D. J.Gordon et al. (1986) Circulation 74:1217. It has been proposed that a 1%increase in the concentration of HDL would lead to a 3% reduction inrisk for developing clinical atherosclerosis in man. Gordon and Rifkind,supra.

The plasma protein enzyme lecithin-cholesterol acyltransferase (LCAT)catalyzes the transfer of fatty acid from the sn-2 position of lecithinto the free hydroxyl group of cholesterol. J. A. Glomset et al. (1966)J. Lipid Res. 7:638). J. McLean et al. (1986) Proc. Nat'l. Acad. Sci.USA 83:2335-2339 described the cloning and sequence of a human LCATcDNA. J. McLean et al. (1986) Nucl. Acids Res. 14:9397-9406 described acomplete gene sequence for human LCAT.

It was first proposed nearly 30 years ago that the esterificationprocess with this enzyme could be the key step in transferringcholesterol from the tissues of the body to the liver. This process,termed “reverse cholesterol transport” (J. A. Glomset (1968) J. LipidRes. 9:155), was proposed to facilitate the removal of cholesterol fromthe body. However, increases in LCAT were not known to diminish the riskof atherosclerosis.

Various mutations of the LCAT gene are known. Individuals who arehomozygous for a non-functional LCAT mutant have classic LCAT deficiencydisease, characterized by clouding of the cornea, normochromic anemiaand glomeruloscierosis. Mutations in the LCAT gene that result in someresidual LCAT activity lead to Fish Eye disease, characterized byopacity of the cornea and hypoalphalipoproteinemia. H.-G. Klein et al.(1992) J. Clin. Invest. 89:499-506.

Thus, there is a need for compositions and methods for the prophylacticand therapeutic treatment of atherosclerosis and conditions associatedwith LCAT deficiency. This invention satisfies this need by providingcompositions and methods for increasing the serum level of LCATactivity.

SUMMARY OF THE INVENTION

It has been discovered that increasing the level of lecithin-cholesterolacyltransferase activity in a rabbit (which is an accepted model of thedevelopment of atherosclerosis in humans; D. J. Gordon and B. M. Rifkind(1989) N. Engi. J. Med. 321:1311), causes a decrease in the accumulationof cholesterol in the arteries. This discovery is surprising because noprior results had indicated that increasing the level of LCAT activitywould have such an effect. In the rabbit model, increasing the massquantity of human LCAT in the serum by about five times above the normalhuman level also led to significant decreases in total triglycerides, atleast five-fold increases in the amount of high density lipoproteins andabout a seven-fold decrease in the ratio of total cholesterol to highdensity lipoproteins in animals fed a high cholesterol diet. Therefore,increasing LCAT activity in the serum of humans, rabbits and othermammals with similar modes of lipoprotein metabolism is an effectivetreatment against atherosclerosis.

This invention provides methods for treating atherosclerosis in amammalian subject, including humans, comprising the step of increasingthe LCAT activity in the serum of the subject to a level effective todecrease the accumulation of cholesterol in the subject, wherebydecreasing the accumulation of cholesterol provides a treatment foratherosclerosis.

In one aspect, the method involves administering to the subject apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a pharmacologically effective amount of LCAT. In oneembodiment, the composition is administered intravenously. Thisinvention also provides pharmaceutical dosage forms comprising apharmaceutically acceptable carrier and a pharmacologically effectiveamount of LCAT.

In another aspect, the method involves transfecting cells with a nucleicacid comprising a nucleotide sequence coding for expression of LCAT,whereby the transfected cells express LCAT and secrete sufficient LCATinto the serum to increase LCAT to a level effective to decrease theaccumulation of cholesterol. In one embodiment, the method involvestransfecting cells in vivo. In another embodiment, the method involvestransfecting cells ex vivo and administering transfected cells thatexpress and secrete LCAT to the subject in an amount sufficient toincrease LCAT activity to a level effective to decrease the accumulationof cholesterol.

In another aspect, the methods involve administering a drug thatup-regulates the endogenous production of LCAT in the subject.

In another aspect, the method involves increasing both the serum LCATactivity and the level of Apo A-I in the serum to an amount effective todecrease accumulation of cholesterol. The invention also providesvectors comprising a nucleic acid that comprises expression controlsequences operatively linked to a sequence that codes for the expressionof LCAT and expression control sequences operatively linked to asequence that codes for the expression of an Apo A-I.

This invention also provides methods for treating an LCAT deficiencycondition in a mammalian subject comprising increasing the LCAT activityin the serum of the subject to a therapeutically effective level. Thecondition can be Fish Eye Syndrome or Classic LCAT Deficiency Syndrome.

This invention also provides non-human mammals transgenic for LCAThaving an absolute serum LCAT activity of at least 1000 nmol/ml/hr.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a comparison of the atherosclerosis in control andtransgenic rabbits over-expressing human LCAT by quantitativeplanimetry. The aortas of LCAT transgenic and control male rabbits, feda 0.3% cholesterol-chow diet for 17 weeks, were harvested and stainedwith Sudan IV. The percent of the surface area that stained wasdetermined by planimetry of the digitized image. J. F. Cornhill et al.,(1985) Arteriosclerosis, 5:415. Top panel: Planimetry of aortas oftransgenic rabbits (n=12). Middle panel: Planimetry of aortas of controlrabbits (n=10). The compilations of the images from the study groups aresummarized for transgenic and control rabbits. Bottom panel: Gradedshades of the probability of distribution.

FIG. 2. Photographs of cross sections of aorta from control andtransgenic rabbits fed a high cholesterol diet. A 1 mm section was takenat the descending thoracic aorta at the same position for each aorta,stained with PAS, and the degree of foam cell accumulation in the intimaof the controls was compared to the lack of intimal cell formation orchange in intimal cell thickness in the transgenic aortae. Left handphotograph: Cross section of aorta of control rabbit. Right handphotograph: Cross section of aorta from transgenic rabbit.

FIG. 3 shows the extent of intimal cellular proliferation as shown inFIG. 2, quantitated using a ratio of the intima to media. A. V.Chobanian et al., (1989) Hypertension 14:203. Left hand graph:Quantitative assessment of the intimal/media ratio (p<0.003). Right handgraph: Quantitative assessment of the percent of surface area (p<0.009).In both cases, the quantitative assessment was significantly lower inthe transgenic LCAT rabbits than in controls.

FIG. 4 shows the significant (<0.05) bivariate Pearson correlations ofpost-diet variables with aortic atherosclerosis in control andtransgenic rabbits. The intima/media ratio correlated well with theplanimetry assessment of atherosclerosis in both the control group andthe entire study. FIG. 4A: Control group, showing % surface area, FIG.4B: control group, showing LCAT activity in nmol/ml/h, FIG. 4C: controlgroup, showing non-HDL cholesterol, in mg/dl; FIG. 4D: control group,showing ratio of total cholesterol to HDL cholesterol. FIG. 4E: entirestudy group (controls and transgenics), showing % surface area, FIG. 4F:entire group, showing LCAT activity in nmol/mlh, FIG. 4G: entire group,showing non-HDL cholesterol, in mg/dl; FIG. 4H: entire group, showingratio of total cholesterol to HDL cholesterol. The intima/media ratiowas inversely correlated with the severity of atherosclerosis (see FIGS.4B and 4F) and positively correlated with the non-HDL cholesterol (seeFIGS. 4C and 4G) and total cholesterol/HDL cholesterol (TC/HDL) (seeFIGS. 4D and 4H).

FIG. 5 depicts the nucleotide sequence and deduced amino acid sequenceof a genomic clone encoding a human LCAT (SEQ ID NO:1). FIG. 5A:Nucleotide sequence of nucleotides 1-1092, deduced amino acid sequenceand exon start and start site. FIG. 5B: Nucleotide sequence ofnucleotides 1093-2100, deduced amino acid sequence, and exon start andstop sites. FIG. 5C: Nucleotide sequence of nucleotides 2101-3108,deduced amino acid sequence, and exon start and stop sites. FIG. 5D:Nucleotide sequence of nucleotides 3109-4368, deduced amino acidsequence, and exon start and stop sites. FIG. 5E: Nucleotide sequence ofnucleotides 4368-5292, deduced amino acid sequence, and exon start andstop sites. FIG. 5F: Nucleotide sequence of nucleotides 5293-6552,deduced amino acid sequence, and exon start and stop sites. FIG. 5G:Nucleotide sequence of nucleotides 6553-6901 FIG. 5F: Nucleotidesequence of nucleotides 5293-6552, deduced amino acid sequence, and exonstart and stop sites.

DETAILED DESCRIPTION OF THE INVENTION

Since the process of accumulating cholesterol in the arteries leads toatherosclerosis and its clinical sequelae, for example, ischemic heartdisease and heart attack, stroke and peripheral vascular disease,slowing or reversing the process of cholesterol accumulation iseffective in the prevention or treatment of atherosclerosis. Thus, thisinvention provides compositions and methods for decreasing (i.e.,slowing or reversing) the accumulation of cholesterol in the arteries ofa mammalian subject by increasing the level of lecithin-cholesterolacyltransferase (“LCAT”) activity in the serum of the subject. Inanother aspect, this invention provides methods of maintaining a ratioof total serum cholesterol to serum high density lipoproteins in amammal at below five:one, considered to be a profile of average risk forheart disease. (W. P. Castelli et al. (1986) JAMA 256:2835.) In anotheraspect, this invention provides methods for prophylactically ortherapeutically treating atherosclerosis in a mammalian subject, inparticular, a human subject, by increasing the activity of LCAT in theserum of the subject to a rate effective to decrease the accumulation ofcholesterol of the subject, in particular in the arteries of thesubject. In another embodiment, this invention provides methods fortreating conditions involving LCAT deficiencies, such as Fish EyeSyndrome and Classic LCAT Deficiency Disease. The methods involveincreasing the LCAT activity in the serum to a level sufficient toameliorate the condition and, preferably, increasing it to at leastnormal levels.

As used herein, “lecithin-cholesterol acyltransferase,” or “LCAT,”refers to a glycoprotein enzyme that can be found naturally in the bloodserum of mammals, including humans, that catalyzes the synthesis ofcholesterol esters and lysolecithin from phosphatidylcholine andunesterified cholesterol present in plasma lipoproteins. The enzyme isnaturally produced primarily by the liver. Genomic DNA encoding a humanLCAT of 416 amino acids has been isolated. Its nucleotide sequence anddeduced amino acid sequence are provided in FIG. 5 (SEQ ID NO:1). Thenucleotide and deduced amino acid sequence of an LCAT from mouse isdescribed in C. H. Warden et al. (1989) J. Biol. Chem. 264:21573-81. Anymammalian LCAT or enzymatically active allelic variation of it is usefulin the methods of this invention, as are other variants, includingfragments of the enzyme that possess the enzymatic activity describedabove. An “allelic variation” in the context of a polynucleotide or agene is an alternative form (allele) of a gene that exists in more thanone form in the population. At the polypeptide level, “allelic variants”generally differ from one another by only one, or at most, a few aminoacid substitutions. A “species variation” of a polynucleotide or apolypeptide is one in which the variation is naturally occurring amongdifferent species of an organism. A polypeptide “fragment” or “segment”is a stretch of amino acid residues of at least about 6 contiguous aminoacids from a particular sequence, more typically at least about 12 ammoacids.

The amount of LCAT or LCAT activity in the serum can be determined invarious ways. The mass of LCAT can be determined by a competitive doubleantibody radioimmunoassay. Routine methods also are known for measuringabsolute LCAT activity in the serum and for measuring the moreinformative cholesterol esterification rate. See, e.g., J. J. Albers etal. Methods in Enzymol. 129:763-783 (1986) and M. P. T. Gillett and J.S. Owens, Chapter 7b, pp. 187-201, in Lipoprotein Analysis—A PracticalApproach, C. A. Converse and E. R. Skinner, eds. LCAT activity can bedetermined by measuring the conversion of radiolabeled cholesterol tocholesteryl ester after incubation of the enzyme and radiolabeledlecithin-cholesterol liposome substrates containing Apo A-I. Endogenouscholesterol esterification rate can be determined by measuring the rateof conversion of labeled cholesterol to cholesteryl ester afterincubation of fresh plasma that is labeled with a trace amount ofradioactive cholesterol by equilibration with a a [¹⁴C]cholesterol-albumin mixture at 4° C. A more detailed protocol isprovided in the Examples. The endogenous cholesterol esterification rateis a better measure of the therapeutic LCAT activity because it reflectsnot only the amount of LCAT activity present in the serum, but also thenature and amount of substrate and cofactors in the plasma. Thus, thecholesterol esterification rate is not necessarily proportional toeither mass of LCAT or absolute LCAT activity.

As used herein, “mammalian subject” refers to an individual of anymammalian species that develops signs of atherosclerosis, includinghumans. These signs or indicators include, for example, the developmentof cholesterol plaques in the arteries and calcification, the extent ofwhich can be determined by Sudan IV staining, or the development of foamcells in an artery. Atherosclerosis also is characterized by a narrowingof the arteries detected by, for example, coronary angioplasty,ultrasound and ultrafast CT. Preferred mammalian subjects for LCATtreatment of atherosclerosis include those whose mode of lipoproteinmetabolism is similar to humans and rabbits, such as non-human primates.

Prophylactic and Therapeutic Treatments

As used herein the term “treatment” refers to both prophylactic andtherapeutic treatment. For example, prophylactic treatment can beadministered to those subjects at risk for developing atherosclerosis.One risk factor is an atherogenic lipoprotein profile. For example, aratio of serum cholesterol to high density lipoproteins of above 5:1indicates a higher than average risk of developing atherosclerosis.Other factors indicating increased risk for atherosclerosis include aserum cholesterol level of above about 240 mg/dl; a high densitylipoprotein level below about 35 mg/dl; and a low density lipoproteinlevel above about 190 mg/dl. Therapeutically, the treatment of thisinvention can be administered to individuals already suffering fromatherosclerosis or a disease associated with it.

The level of serum LCAT activity effective to decrease accumulation ofcholesterol depends on several factors, including the species, themanner of administration, the general health of the subject, the desiredresult (e.g., prophylaxis or therapeutic treatment) and the judgment ofthe prescribing physician. For example, the practitioner may decide whatrisk levels for heart disease indicate prophylactic treatment, and whattarget level of LCAT is indicated for the treatment of a person alreadysuffering from atherosclerosis. LCAT levels sufficient to measurablydecrease the rate of accumulation of cholesterol are at least aboutone-and-a-half times the normal cholesterol esterification rate for themammalian species and, more preferably, at least about two times, atleast about five times, at least about ten times, at least about fifteentimes or at least about twenty times.

In humans, the normal cholesterol esterification rate ranges from about60 nmol/ml/hr to about 130 nmol/ml/hr. The effective treatment ofatherosclerosis in humans can involve increasing the level of serum LCATactivity to achieve a cholesterol esterification rate of at least 200nmol/ml/hr, at least 250 nmol/ml/hr, at least 500 nmol/ml/hr, at least1000 nmol/ml/hr or at least 2000 nmol/ml/hr. Increasing the mass of LCATin the serum results in an increase in cholesterol esterification rates.Normally, humans have about 5 μg LCAT per ml serum. Thus, increasingLCAT to at least 10 μg/ml of serum can increase the cholesterolesterification rate to anti-atherosclerotic levels. Increasing LCAT toat least 15 μg/ml of serum, at least 25 μg/ml of serum, at least 50μg/ml of serum or at least 100 μg/ml of serum also can achieve theseends.

Methods for Increasing Serum Levels of LCAT

The serum level of LCAT can be increased by any means available. Thisincludes, without limitation, direct administration of the LCAT enzyme,expression of LCAT through gene therapy and the up-regulation ofendogenous LCAT through the administration of drugs.

1. Administration of LCAT Enzyme

The level of LCAT activity in a subject can be increased byadministering to the subject a pharmaceutical composition comprising apharmaceutically acceptable carrier and a pharmacologically effectiveamount LCAT. A pharmacologically effective amount of LCAT is an amounteffective to decrease the rate of accumulation of cholesterol in asubject. As used herein, the term “pharmaceutical composition” refers toa composition suitable for pharmaceutical use in a mammal. Thus,pharmaceutical compositions are relatively non-toxic to the animal towhom the composition is administered.

“Pharmaceutically acceptable carrier” encompasses any of the standardpharmaceutical carriers, buffers and excipients, such as a phosphatebuffered saline solution, water, and emulsions. Suitable pharmaceuticalcarriers and their formulations are described in Martin, Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton 1995).Liquids are the preferred pharmaceutical carriers for the preparation ofthe pharmaceutical compositions of this invention.

The pharmaceutical compositions are intended for all well known forms ofadministration and, in particular, parenteral administration forprophylactic and/or therapeutic treatment. Local administration, such astransdermally, also is contemplated. The pharmaceutical compositions canbe administered in a variety of unit dosage forms depending upon themethod of administration. For example, unit dosage forms for parenteraladministration include unit doses of injectable solutions.

LCAT can be administered by any means known in the art for delivery ofproteins. However, systemic administration by injection is preferred.This includes intramuscular, intravenous, intraperitoneal, andsubcutaneous injection. Injection can be automated by, for example, aprogrammable pump. Thus, this invention provides compositions forintravenous administration which comprise a solution of LCAT dissolvedor suspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., water, buffered water,0.4% saline, and the like. For instance, phosphate buffered saline (PBS)is particularly suitable for administration of LCAT protein. Thesecompositions can be sterilized by conventional, well-known sterilizationtechniques, or can be sterile filtered. Additives can also includeadditional active ingredients such as bactericidal agents, orstabilizers. The resulting aqueous solutions can be packaged for use asis, or lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH-adjusting and bufferingagents, tonicity-adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

LCAT also can be systemically administered transmucosally ortransdermally. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrations are generally known in the art, andinclude, for example, for transmucosal administration, bile salts andfusidic acid derivatives. In addition, detergents can be used tofacilitate permeation. Transmucosal administration can be through nasalsprays, for example, or using suppositories.

The form, amounts and timing of administration generally are a matterfor determination by the practitioner. In one embodiment, thepharmaceutical composition is delivered as a unit dosage form. It isestimated that about 40 mg LCAT suffices to increase the amount of serumLCAT in a human about two-fold. Accordingly, a unit dosage form cancontain about 10 mg to about 1000 mg or about 40 mg to about 200 mg. Incertain embodiments, the dosage form has about 100 mg, or about 500 mgLCAT. Depending on the target level of LCAT to be maintained and timingof delivery, small doses can be administered frequently, or large dosescan be administered less frequently.

The invention contemplates several sources of LCAT for incorporationinto pharmaceutical compositions. LCAT can be isolated from plasma. Amethod for isolating LCAT from human serum is described in the Examples.

Alternatively, LCAT can be recombinant LCAT and, more particularly,recombinant human LCAT. J. McLean et al. (1986) Proc. Nat'l. Acad. Sci.USA 83:2335-2339 describes the cloning and sequence of a human LCATcDNA. J. McLean et al. (1986) Nucl. Acids Res. 14:9397-9406 describes acomplete gene sequence for human LCAT. (See FIG. 5.) J. S. Hill et al.(1993) J. Lipid Res., 34:1245-1251 describes the expression ofrecombinant LCAT. Methods of preparing expression vectors are describedin further detail below. These vectors can be used to expressrecombinant LCAT in a variety of expression systems including mammalian,insect and bacterial systems. The unicellular hosts may be prokaryoticor eukaryotic and include, for example, E. coli, yeast, insect cells,COS cells or Chinese Hamster Ovary (“CHO”) cells. A method forexpressing LCAT in COS-7 cells is described in J. McLean (1986) Proc.Nat'L Acad. Sci., USA., 83:2335-2339. Expression control sequences canbe chosen as appropriate. Recombinant LCAT can then be purified by e.g.,the method described in the Examples.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and virusesthat incorporate the recombinant polynucleotide. The construction ofexpression vectors and the expression of genes in transfected cellsinvolves the use of molecular cloning techniques also well known in theart. Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., (1989) and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., (CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc.

2. Gene Therapy

In one aspect, the level of LCAT activity is increased through the useof gene therapy. As used herein, “gene therapy” refers to the transferand, preferably, stable integration of new genetic information intocells in a subject. Methods of increasing LCAT activity levels by genetherapy involve transfecting cells with a nucleic acid that includes anucleic acid sequence coding for expression of LCAT. The transfectedcells express LCAT and secrete it into the serum of the subject. Thecells are transfected in sufficient number or for such high expressionof LCAT that they increase the amount of LCAT to a level effective todecrease the accumulation of cholesterol. Genes encoding LCAT areintroduced into the subject in one of two ways. In one embodiment, thegenes are introduced into cells of the individual in vivo by means ofexpression vectors. In another embodiment, the genes are introduced intocells ex vivo, and transfected cells that express and secrete LCAT areadministered to the subject.

In in vivo approaches, liver cells are useful targets for transfection.Liver cells produce LCAT, so they possess the processing machinery formaking the enzyme recombinantly. Furthermore, vectors injected into theblood stream quickly pass through the liver, so liver cells are quicklyexposed to the vectors. Hematopoietic stem cells also are useful targetsfor gene therapy because they multiply rapidly, thereby creating morecells capable of producing LCAT. vivo approaches also are attractivebecause they allow more control over the transfection process. Forexample, transfected cells can be tested and the ones which express LCATin the highest amounts can be selected. Hematopoietic stem cells can betaken from the subject, transfected ex vivo and reintroduced into thesubject. Therefore, in one embodiment, the cells are cells from thesubject. While allografts can be useful, syngeneic grafts are mostuseful since they are least likely to elicit a host-vs-graft response.

Methods of transfecting genes into mammalian cells, either in vivo andex vivo, and obtaining their expression are well known to the art. Theseinclude, for example, transfecting cells with the nucleic acid by meansof nucleic acid vectors, such as viral vectors (including, e.g.,retroviral vectors, adenoviral vectors, adeno-associated viral vectors,hepatitis viral vectors, vaccinia viral vectors and herpes viralvectors), plasmid vectors, cosmid vectors, microencapsulation vectors(e.g., cationic or uncharged liposomal microspheres); microinjection;electroporation; chromosome transfer; calcium precipitation; orbiolistic injection (e.g., attaching DNA to a particle, such as a goldbead, and propelling it into a cell). See also, e.g., Methods inEnzymology, vol. 185, Academic Press, Inc., San Diego, Calif. (D. V.Goeddel, ed.) (1990) or M. Kriegler, Gene Transfer and Expression—ALaboratory Manual, Stockton Press, New York, N.Y., (1990).

Viral vectors are particularly useful for gene therapy. Methods forconstructing and using viral vectors are known in the art and arereviewed, for example, in Miller and Rosman (1992) Biotechniques,7:980-990. The targeting specificity of viral vectors can be utilized totarget predetermined cell types and introduce a nucleic acid moleculeinto the cell. Thus, the viral vector selected will depend, in part, onthe cell type to be targeted. For example, hepatocytes, which normallyproduce LCAT, are an attractive target for transfection. In addition, aviral vector can be made tissue-specific by incorporating atissue-specific promoter or enhancer into the vector.

Adenoviruses are useful vectors for the transfer of genes into cells.Liver cells have an adenovirus receptor. Therefore, upon intravenousinjection of a recombinant adenovirus, about 95% of the viruses willselectively infect liver cells. Replication-defective adenoviral vectorscan be prepared by deletion of sequences spanning E1A, E1B, and aportion of the E3 region, impairing the ability of this virus toreplicate or transform non-permissive cells. See, e.g., Hurwitz et al.(1985) Proc. Natl. Acad. Sci. U.S.A. 82:163-167. The earlyenhancer/promoter of the cytomegalovirus can be used to drivetranscription of an inserted LCAT gene with an SV40 polyadenylationsequence cloned downstream from this reporter. High titer recombinantadenovirus can be prepared by amplification in LE293 cells usingestablished methods. Virus can be purified from cell lysates by cesiumchloride gradient ultra-centrifugation followed by de-salting onSephadex G-50 (Sigma Biochemicals, St. Louis Mo.) column in PBS.Purified virus then can be used for injection into the subject.Adenoviral vectors in which the E4 region has been deleted also areattractive as second-generation adenoviral vectors that have prolongedexpression and less possible immunogenicity.

Adenoassociated virus (AAV), is a single-stranded, DNA parvovirus. AAVvectors have several advantages which make them desirable as genedelivery systems. They have no known mode of pathogenesis and 80% ofpeople in the United States are currently seropositive for AAV. Ostroveet al. (1981) Virology 113:521; Cukor et al., in The Parvoviruses (ed.Berns, Plenum, N.Y., 1984). AAV virions are resistant to physicaltreatments, such as sonication and heat inactivation not tolerated byother viruses during purification. Samulski et al. (1989) J. Virol.63:3822-3828. Unlike retroviruses, infection and/or transduction ofnon-dividing cells is possible. Like retroviruses, AAV integrates intothe host cell genome upon infection. Kotin et al. (1990) Proc. Natl.Acad. Sci. USA 87:2211-2215; Samulski et al. (1991) EMBO J.10:3941-3950. However, unlike retroviruses, AAV preferentiallyintegrates at a specific chromosomal site (19q13.3). At this site, AAVdoes not cause any significant alteration in the growth properties ormorphological characteristics of human cells. Furthermore, integrationof AAV into the cellular genome can occur in non-proliferating cells.Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996. AAV also possess areasonably large cloning capacity, slightly more than half that ofretroviral vectors. Recombinant AAV vectors contain no endogenouspromoter activity, allowing for specific promoter choices to be madedepending on target cell type, without regard for complications arisingfrom endogenous viral promoters. Unlike retroviral vectors, packaged AAVvectors can be concentrated so that multiplicities of infectionexceeding 1.0 can be used in transduction experiments. This means thatvirtually 100% of the targets in a culture could be transduced,obviating the need for a selection step.

AAV is a defective virus that grows only in cells in which certainfunctions are provided by a co-infecting helper virus such asadenoviruses and herpesviruses. Infection of cells with AAV in theabsence of helper functions results in integration of AAV into the hostcell genome without replication. The AAV genome has two copies of a145-nucleotide-long ITR (inverted terminal repeat), one at each end.Srivastava et al. (1983) J. Virol. 45:555-564. The ITR sequences providean origin of replication and also mediate integration and excision ofthe AAV genome from the cell genome.

The sequence between the ITRs of about 4470 nucleotides contains twoopen reading frames for rep and cap genes. Hermonat et al. (1984)Virology 51:329-339. The cap gene encodes capsid proteins. The rep genneencodes proteins known to be required for replication. Rep⁻ vectors mayshow reduced preference for site-specific integration into chromosome19. However, the overall integration frequency of rep⁻ vectors is about80-fold higher integration frequency than comparable rep⁺ vectors,suggesting rep inhibits rather than facilitates integration. McLaughlinet al. (1988) J. Vitrol. 62:1963-1973.

In recombinant AAV, all protein coding sequences (such as cap, lip andrep) can be replaced by the LCAT encoding sequence. Recombinant AAV isreplicated by co-transfecting a cell bearing the AAV vector carrying thegene of interest, together with a helper AAV plasmid that expresses allof the essential AAV genes, into adenovirus- or herpes-infected cells,which supply additional helper functions necessary for AAV replicationand the production of new viral particles.

A further method has been proposed in which a recombinant vectorcontaining AAV ITR sequences but lacking all other AAV sequences issurrounded by cationic lipids and introduced into a cell by lipofection.Phillip et al., WO 95/07995.

In the case of non-infectious viral vectors, the helper virus genome isusually mutated to destroy the viral packaging signal required toencapsulate the nucleic acid into viral particles. However, the helpervirus retains structural genes required to package a co-introducedrecombinant virus containing a gene of interest. Without a packagingsignal, viral particles will not contain a genome and, thus, cannotproceed through subsequent rounds of infection.

Retroviral vectors can be used for in vivo or ex vivo targeting andtherapy procedures. Retroviral vectors can be constructed either tofunction as infectious particles or to undergo only a single initialround of infection. In the former case, the genome of the virus ismodified so that it maintains the necessary genes, regulatory sequencesand packaging signals to synthesize new viral proteins and RNA. However,genes conferring oncogenic potential of these viruses are destroyed.After the viral proteins are synthesized, the host cell packages the RNAinto new viral particles, which can undergo further rounds of infection.The viral genome also is engineered to encode and express the desiredrecombinant protein. Improved methods of transfection of peripheralblood lymphocytes by retroviral vectors are described in Yang et al.(1995) Nature Medicine 1(9):890-893 and Bunnell et al. (1995) Proc.Acad. Nat'l Sci. USA 92:7739-7743.

A retroviral packaging cell line such, as PA317 (American Type CultureCollection, Bethesda, Md., accession number CRL 9078) can be used tocreate infective amphotrophic retroviral vectors. The retroviralplasmid, pLNCX (D. Miller and G. Rosman (1989) Biotechniques 7:980) cancontain the expression control sequence operatively linked to thenucleic acid sequence to be expressed at “X”. That plasmid contains aMaloney murine leukemia virus LTR promoter/enhancer (L); neomycinresistance gene encoding neomycin phosphotransferase (N); a humancytomegalic virus LTR/enhancer (C) and the coding gene to be expressed(X). The LCAT gene is inserted by standard techniques at a pre-existingcloning site by replacement of the phosphotransferase gene for neomycinresistance for one encoding a phosphotransferase of hygromycinresistance.

Retroviruses have been the preferred vehicle for gene delivery intohuman hematopoietic stem cells because of their high efficiency of genetransfer and the co-linear and stable integration of the transferredgenes into chromosomal DNA. However, genes from retroviruses canintegrate into the host cell chromosome only if the cell is activelydividing.

Nucleic acids used to transfect cells with the LCAT gene generally willbe in the form of an expression vector including an expression controlsequence operatively linked to a nucleotide sequence coding forexpression of LCAT. The term “expression control sequence” refers to anucleotide sequence in a polynucleotide that regulates the expression(transcription and/or translation) of a nucleotide sequence operativelylinked thereto. The term “operatively linked” refers to a functionalrelationship between two parts in which the activity of one part (e.g.,the ability to regulate transcription) results in an action on the otherpart (e.g., transcription of the sequence). Expression control sequencescan include, for example and without limitation, sequences of promoters(e.g., inducible or constitutive), enhancers, transcription terminators,a start codon (i.e., ATG), splicing signals for introns, and stopcodons.

Appropriate expression control sequences for mammalian cells include,for example, the SV40 promoter, the RSV (Rous sarcoma virus) promoter,the CMV (cytomegalovirus) promoter and the metallothionein promoter. TheCMV promoter is preferred for transient expression systems. Themetallothionein promoter is preferred for stable expression systems.Promoters can be constitutive or can be tissue-specific for the targetcell type, e.g., specific for hepatocytes. Expression control sequencesproviding high levels of expression are preferable for producing LCAT inquantities sufficient to cause a decrease in the accumulation ofcholesterol.

As used, the term “coding for expression of LCAT” refers to sequencesthat, upon transcription and translation of mRNA, produce polypeptidesequences of the LCAT enzyme. Thus, for example, cDNA, genomic DNA withintrons removed upon transcription and processing into MRNA, anddegenerate sequences encoding LCAT, all code for expression of LCAT. DNAsequences coding for expression of LCAT can be obtained by any methodsknown in the art. For example, coding sequences can be prepared bychemical synthesis. Also, PCR primers can be devised using the sequencesof LCAT provided herein and cDNA or genomic DNA can be isolated byamplification. The following polynucleotides are useful as primers tocreate probes for isolating from a library a cDNA or genomic cloneencoding a human LCAT.

1. Probe I: a 340 bp 5′ LCAT exon 1 probe

Sense oligo: 5′ GGC TCC CTG AGG CTG TGC CCC TTT 3′

(SEQ ID NO:2)

Antisense oligo: 5′ TGG CGT GGT GCA TCA GGG GCC TGG 3′

(SEQ ID NO:3)

2. Probe II: a 380 bp 3′ LCAT probe

Sense oligo: 5° CTG GTG TGG AAG TAT ACT GCT TTT 3′

(SEQ ID NO:4)

Antisense oligo: 5° CTT CAA CCT GAA ACA TAG CCA TCA 3′

(SEQ ID NO:5)

The technique of “polymerase chain reaction,” or “PCR,” as used hereingenerally refers to a procedure wherein minute amounts of a specificpiece of nucleic acid, RNA and/or DNA, are amplified as described inU.S. Pat. No. 4,683,195 issued Jul. 28, 1987. Generally, sequenceinformation from the ends of the region of interest or beyond needs tobe available, such that oligonucleotide primers can be designed; theseprimers will be identical or similar in sequence to opposite strands onthe template to be amplified. The 5′ terminal nucleotides of the twoprimers may coincide with the ends of the amplified material. PCR can beused to amplify specific RNA sequences, specific DNA sequences fromtotal genomic DNA, and cDNA transcribed from total cellular RNA,bacteriophage or plasmid sequences, etc. See, generally, Mullis et al.(1987) Cold Spring Harbor Symp. Quant. Biol. 51:263; Erlich, ed., PCRTechnology, (Stockton Press, N.Y., 1989). As used herein, PCR isconsidered to be one, but not the only, example of a nucleic acidpolymerase reaction method for amplifying a nucleic acid test sample,comprising the use of a known nucleic acid (DNA or RNA) as a primer.

An “isolated polynucleotide” is a polynucleotide, e.g., an RNA, DNA, ora mixed polymer, which is substantially separated from other DNAsequences which naturally accompany a native human sequence, e.g.,ribosomes, polymerases, and many other human genome sequences. The termembraces a polynucleotide sequence which has been removed from itsnaturally occurring environment, and includes recombinant or cloned DNAisolates and chemically synthesized analogues or analogues biologicallysynthesized by heterologous systems. A substantially pure moleculeincludes isolated forms of the molecule. An “isolated polypeptide” orprotein carries a similar meaning with the polypeptide or protein beingsubstantially separated from any cellular contaminants and componentsnaturally associated with the protein in vivo, so that it is thepredominant macromolecular species in the composition.

Some of the disadvantages stemming from the use of viral vectors areavoided by transfecting a DNA fragment into cells nonbiologically, forexample, by lipofection, chemical transformation, electroporation orbiolistic injection. In these approaches, ample amounts of pure DNA canbe prepared for transfections, and much larger fragments can beaccommodated. Such approaches are preferred for cells that can betemporarily removed from the body.

The term “recombinant” or “recombinant DNA molecule” refers to a nucleicacid sequence which is not naturally occurring, or is made by theartificial combination of two otherwise separated segments of sequence.By “recombinantly produced” is meant artificial combination oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques. Such is usually done to replace a codon with aredundant codon encoding the same or a conservative amino acid, whiletypically introducing or removing a sequence recognition site.Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the common natural forms.Restriction enzyme recognition sites are often the target of suchartificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design.“Recombinant DNA molecules” include cloning and expression vectors. A“fragment” of a polynucleotide is a stretch of at least about 18nucleotides, more typically at least about 40 nucleotides.

3. Up-Regulation of Endogenous LCAT

Another method for increasing the serum level of LCAT activity isthrough the administration of drugs that up-regulate the endogenousproduction of LCAT in the subject. Androgens already are known todown-regulate the expression of LCAT. J. J. Albers et al. Biochim.Biophys. Acta 795:293-296 (1984). Also, LCAT levels are higher infemales than in males. J. J. Albers et al. Atherosclerosis 43:369-379(1983). It is expected that estrogens and estrogen analogs up-regulateexpression of LCAT. The amounts of such drugs to be administered can bedetermined empirically by the practitioner.

Candidate drugs can be identified by screening compounds for the abilityto increase LCAT expression in animal models or in in vitro models. Inanimal models, the candidate drug can be administered to an animal todetermine the effect on LCAT activity in the animal. Alternatively,after administration, the animal can be examined to determine the effectof the drug on the development of atherosclerosis.

Candidate drugs can be identified by screening compounds for the abilityto increase LCAT activity in vitro. In one method, the compound istested for its effect on the ability of LCAT to cause esterification ofcholesterol to cholesteryl ester in an LCAT activity assay such as thatdescribed above.

Further Methods of Treatment

The metabolism of cholesterol involves both the loading of cholesterolonto HDL particles through esterification by LCAT and the transport ofcholesterol to the liver by HDL. It is known that increasing Apo A-Idecreases the risk of heart disease. A. C. Liu et al. (1994) J. LipidRes. 35:2263-2267 and C. Paszty et al. (1994) J. Clin. Invest.94:899-903. Certain versions of Apo A-I, such as Apo A-I milano, areparticularly protective. C. R. Sirtori et al. (1995) Atherosclerosis Xpp. 50-55, Elsevier Science, NY and S. Ameli et al. (1994) Circulation90:1935-41. Furthermore, Apo A-I is a cofactor of LCAT activity. C. J.Fielding et al. (1972) Biochim Biophys. Acta 270:513-518. Accordingly,increasing the activity of LCAT in the serum of the subject combinedwith increasing the transport of cholesterol to the liver by increasingserum HDL or by providing HDL with better cholesterol loadingcharacteristics produces a synergistic effect for decreasing theaccumulation of cholesterol in the arteries of a subject. In oneembodiment, the invention involves increasing both LCAT and the level ofApo A-I in the serum of a subject to an amount effective to decreaseaccumulation of cholesterol. The amount of Apo A-I in the serum of ahuman normally is about 130 mg/dl. In the methods of this invention,levels of Apo A-I can be increased to at least 150 mg/dl or at least 300mg/dl. In one embodiment, the levels of both Apo A-I and Apo A-I milanoare increased. Apo A-I milano also is increased to at least 150 mg/dl orat least 300 mg/dl. Methods for increasing the amount of Apo A-I includeany of already described for increasing the levels of LCAT. Methods ofproducing Apo A-I are described in U.S. Pat. No. 4,943,527 (Protter etal.).

This invention also provides expression vectors comprising a nucleicacid that includes expression control sequences operatively linked to asequence that codes for the expression of LCAT. In another embodimentthis invention provides expression vectors comprising a nucleic acidthat includes expression control sequences operatively linked to asequence that codes for the expression of LCAT and expression controlsequences operatively linked to a sequence that codes for the expressionof an Apo A-I protein, including, e.g., Apo A-I milano.

Treatment of Conditions Related to LCAT Deficiency

In another aspect, this invention provides methods for treatingconditions stemming from a deficiency of LCAT activity, such as ClassicLCAT Deficiency Syndrome, in which LCAT is absent, and Fish EyeSyndrome, in which the individual has only partial, residual LCATactivity levels. The methods involve increasing serum LCAT activitylevels in such individuals to a therapeutically effective level.Increasing LCAT serum levels to about normal levels, i.e., to achieve acholesterol esterification rate at least 60 nmol/ml/hr or at least 130mnol/ml/hr, are therapeutically effective. Also, LCAT can be increasedto achieve a cholesterol esterification rate above normal levels, e.g.,rates of about 200 nmol/ml/hr, 300 nmol/mI/hr, 500 nmoUml/hr or 1000nmol/ml/hr. Increasing the mass of LCAT in the serum to about 5 μg/ml,or higher, e.g., about 10 μg/ml, about 20 μg/ml, about 50 μg/ml or about100 μg/ml, also achieves these results. LCAT levels can be increased byany of the methods described above.

Transgenic Non-Human Mammals

This invention also provides non-human mammals transgenic for LCAT whoseserum has a cholesterol esterification rate of at least 1.5 times or atleast two times the normal level, or an absolute LCAT serum activity ofat least 1000 nmol/ml/hr and, preferably, at least 1500 nmol/ml/hr.These animals are useful in the production of healthier livestock, inthe study of atherosclerosis and as screens for compounds that increaseor decrease the accumulation of cholesterol. As used herein, “mammaltransgenic for LCAT” refers to a mammal whose germ cells (i.e., oocytesor sperm), at least, comprise a recombinant nucleic acid moleculecomprising expression control sequences operatively linked to a nucleicacid sequence coding for expression of LCAT. In one embodiment, theexpression control sequences are not naturally found operatively linkedto LCAT. In a preferred embodiment, the recombinant nucleic acidcomprises a non-native LCAT coding sequence, i.e., an LCAT sequence thatthe species does not produce in nature. In one embodiment, the LCAT is ahuman LCAT. Particularly useful transgenic mammals of this inventioninclude rabbits and rodents such as mice. Transgenic non-human mammalsof this invention can be produced as described in the Examples.

EXAMPLES Determination of Endogenous Cholesterol Esterification Rate

J. J. Albers et al. (1986) Methods in Enzymology 129:763-783 describethe following method for determining endogenous cholesterolesterification rate. For every 25 assays, the enzymatic color reagent ismade fresh daily by mixing 0.1 ml of 1 mg cholesterol oxidase/mlsolution. 1 ml of 2 mg peroxidase/ml solution, 1 ml of 20 mg4-aminoantipyrine/ml solution, 1 ml of 50 mg 2.4-dibromophenol/mlsolution, 3.75 ml of 2% sodium cholate solution, and 30.65 ml of assaybuffer. Forty microliters of fresh plasma is pipetted into glass tubes,in septuplicate for both control and test samples. At zero time, 20 μlof 150 mM iodoacetate solution is added to each control sample toinhibit the LCAT reaction, whereas 20 μl of incubation buffer (50 mMphosphate buffer, pH 7.4) is added to each test sample. All samples areincubated at 37° for 40 min. At the end of this incubation, 20 μl of 150mM iodoacetate solution is added to each test sample and 20 μl ofincubation buffer is added to each control sample. Then, 1.5 ml each ofcolor reagent is added to all samples and the mixtures are incubated for10 min at 37°. The absorbance of the assay mixtures are incubated for 10min at 37°. The absorbance of the assay mixtures is measuredspectrophotometrically at a wavelength of 500 nm. Unesterifiedcholesterol in each sample is determined by comparison against the colorof cholesterol standard solution containing 1 to 100 μg of unesterifiedcholesterol in which color is developed in the same manner in the samplesolution. The rate of cholesterol esterification is obtained bysubtraction of the amount of cholesterol in the test samples from thatin the control samples. The rate can be expressed as both fractionalcholesterol esterification rate (percentage decrease in unesterifiedcholesterol/hr) and molar cholesterol esterification rate (nmol decreasein unesterified cholesterol/ml plasma/hr).

Isolation of LCAT

J. J. Albers et al. Methods in Enzymol., 129:763-783 (1986) describesthe following method for isolating LCAT. Plasma is precipitated withdextran sulfate-Mg²⁺ (500 mM MgCl₂) and the plasma is collected. Themixture is added to a Phenyl-Sepharose chromatography column andequilibrated to 10 mM Tris, 140 mM NaCl, and 1 mM EDTA, pH 7.4. LCAT iseluted by washing the column with distilled water. LCAT-containingfractions are dialyzed against 20 mM sodium phosphate, pH 7.1, andpassed through an Affi-Gel Blue column with this buffer. LCAT-containingfractions are isolated. These fractions are equilibrated to 1 mM Tris, 5mM EDTA and 25 mM NaCl, pH 7.4. The mixture is subjected toDEAE-Sepharose chromatography and LCAT is eluted with a linear NaCl-Trisgradient (25 mM NaCl, 1 mM Tris to 200 mM NaCl, 10 mM Tris) containing 5mM EDTA. LCAT-containing fractions are then subject to hydroxylapatitechromatography. LCAT is eluted with a linear phosphate gradient (15 mMto 60 mM), pH 6.9, 150 mM NaCl. The LCAT-containing fractions aresubject to Sephacryl S-200 gel filtration and eluted with 10 mM Tris,140 mM NaCl, 1 mM EDTA, pH 7.4.

Producing Transgenic Animals

Methods for producing transgenic mammals are described in, e.g., C. P.Landel (1991) GATA 8:83-94. Superovulation is induced, by, e.g., firstadministration of pregnant mare's serum, followed by administration ofexogenous gonadotrophin forty-two to forty-eight hours later. Then theanimals are allowed to mate. Fertilized eggs for microinjection are thencollected from superovulated donor females. The females are sacrificedand the oviducts removed. The eggs are removed from the oviduct andwashed in buffer M2, described below in Table 1. The eggs are culturedand separated from cumulus cells in buffer M16, described below. Theeggs are microinjected with purified DNA fragments coding for expressionof LCAT. Intact, microinjected eggs are implanted into the oviducts offoster mothers made pseudopregnant by mating with sterile males.Transgenic non-human animals are identified among the offspring born tothe foster mothers.

The Effect of Increased LCAT on Atherosclerosis in Transgenic Animals

The effect of increased levels of human LCAT on atherosclerosis in therabbit was studied. The rabbit has been the principal animal speciesused to study the development of atherosclerosis caused by dietarycholesterol for more than 80 years (N. Anitschkow and S. Chalatow (1913)Zentralbl. Allg. Path. Anat. 24:1-9). This sensitivity to dietarycholesterol leads to atherosclerosis in the rabbit resembling humanatherosclerotic plaques (M. L. Overturf and D. S. Loose-Mitchell (1992)Curr. Opin. Lipidol. 3:179-185). Rabbit very low density lipoproteinsare similar in their chemical composition, apolipoprotein content, andelectrophoretic mobility with agarose gel electrophoresis to humanvery-low-density lipoproteins. M. J. Chapman (1980) J. Lipid Res.21:789-853. In addition, apolipoprotein B, an apolipoprotein involved inatherogenesis, is evident in rabbit intermediate density lipoproteinsand low density lipoproteins closely resembling that seen in man. Id.Like humans, the rabbit expresses cholesterol ester transfer proteinwhich not only permits the transfer of high density lipoprotein-derivedcholesterol ester to apolipoprotein B-containing lipoprotein particles,but also is likely to play a role in the diet-induced atherosclerosisthai rabbits develop (A. R. Tall (1993) J. Lipid Res. 34:1255-1274).Therefore the rabbit model system affords an excellent means ofdetecting and quantifying the impact of potential therapies to treat andprevent atherosclerosis in humans.

A construct containing the full-length human genomic LCAT gene was usedto generate transgenic animals. B. L. Vaisman et al. (1995) J. Biol.Chem. 270:12269. This construct contained all of the introns and 851 bpof the 5′ and 1134 bp of the 3′ untranslated regions of the human LCATgene. A cosmid library made from human genomic white blood cell DNA wasscreened with a full length human LCAT cDNA probe as described. J.Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, (1989) and B. L. Vaisman et al. (1995) J. Biol.Chem. 270:12269-12275. DNA from identified clones was prepared byalkaline lysis miniprep DNA isolation (J. Sambrook et al., supra) anddigested with the restriction enzyme Sma I (New England Biolabs,Beverly, Mass.). The 6.2 kb Sma I fragment was isolated from agarose gelby gene cleaning (Gene Clean Bio101 Inc., La Jolla, Calif.) followed bysubcloning into bluescript IIKS-(Stratagene, La Jolla, Calif.) togenerate a 9.16 kb plasmid. After amplification in E. coli, plasmid DNAwas isolated by double CsCl banding (S. S. Fojo (1984) Biochem. Biophys.Res. Commun., 124:308-313) followed by extensive dialysis and digestionwith Sma I. The 6.2 kb fragment Sma I fragment was then re-isolated fromagarose gels and gene cleaned (Gene Clean Bio101 Inc., La Jolla,Calif.). The gene cleaned fragment was repurified by CsCl banding,followed by extensive dialysis against injection buffer (10 mM tris pH7.5, 0.1 EDTA) prior to microinjection.

The generation of transgenic rabbits was approved by the Animal Care andUse Committee of the National Heart, Lung, and Blood Institute.Transgenic animals were produced essentially as described above bytransfecting with the Sma I fragment coding for expression of LCAT,above.

In studying the extent of atherosclerosis, animals were sacrificed bypremedication with xylazine (3 mg/pound), ketamine (15 mg/pound), androbinul (0.25 ml/10 pounds) intramuscularly before either isofluraneinhalation anesthesia or euthanasia via intravenous sodiumpentobarbital.

High level of expression of human LCAT led to elevated levels of HDLcholesterol concentrations in the rabbit. J. M. Hoeg et al. (1994)Atherosclerosis 109:11, abstract. This T-1 founder line was expanded forthese studies. A total of 10 LCAT transgenic and 9 control males 5-6moths of age were studied. The human LCAT mass in the transgenic plasmawas 27.25±6.27 μg/ml (mean±SD), which was more than 5-fold the mean LCATmass 5.56+/0.91 μg/ml reported in humans. J. J. Albers et al. (1982)Atherosclerosis 43:369. Expression of human LCAT led to LCAT activity atbaseline in the transgenic rabbits was 15-fold that of controls (Table2). Compared to controls, LCAT transgenic rabbits had a marked increasein total (617%; p<0.001) and HDL cholesterol concentrations (671%;p<0.001).

These rabbits were then fed a 0.3% cholesterol diet (Product number4109000, Ziegler Brothers, Inc., Gardners, Pa.). The high cholesteroldiet led to increases in the control rabbits of both total (19 fold) andespecially non-HDL cholesterol concentrations (127 fold) (Table 2). Incontrast, the plasma concentrations in the LCAT transgenic rabbitsincreased only 2-fold and 11-fold, respectively. The LCAT activity inthe transgenic rabbits on the cholesterol diet remained more than 3-foldthat of the control and led to an increase of HDL cholesterolconcentrations to more than 5-fold that of control rabbits. The totalcholesterol/HDL cholesterol ratio, a sensitive indicator of clinicallydetectable human atherosclerosis (W. Castelli and A. Leaf (1985)Cardiology Clinics 3:171), increased in the control group by-more than12-fold. In contrast, LCAT transgenic rabbit total/HDL ratio rose lessthan 2-fold (Table 2) and remained below the ratio of 5 that provides anaverage risk for atherosclerosis in man. W. P. Castelli et al. (1986)JAMA 256:2835.

These differences in the plasma lipoprotein concentrations betweencontrol and LCAT transgenic rabbits reflected differences in thedevelopment of atherosclerosis. After 17 weeks on the 0.3% cholesteroldiet, the aortae from these animals were harvested, and two methods wereused to quantitate the severity of diet-induced atherosclerosis in theserabbits. Sudan IV staining of the lipid droplets permits thequantitation of the percent of the surface area developing lesions. J.F. Cornhill et al. (1985) Arteriosclerosis 5:415. The probability mapfor aortic lesion development in the transgenic rabbits (FIG. 1) showsonly scattered foci of oil Sudan IV-staining material, whereas controlaortae had substantial staining in the majority of the animals. Theaorta of the control group had 35±7% of the surface covered by plaque.Whereas, in marked contrast, only 5±1% of the aortic surface was coveredby plaque in the LCAT transgenic rabbits (p<0.009, FIGS. 1 and 3,right).

These substantial differences in the atherosclerosis in aortae betweenthese two groups were also evident microscopically (FIG. 2). The intimaof the control rabbits demonstrated foam cell formation, cellularproliferation, and an increase in the ratio of the intima/media to0.40±0.11 (FIG. 3, left). There was virtually no foam cell formation orcellular proliferation in the transgenic rabbits expressing human LCAT(FIG. 2). The 0.03±0.01 intima/media ratio was significantly lower thanthe control (p<0.009; FIG. 3, left). These data establish that theincrease in LCAT activity led to an 85-90% reduction in atherosclerosis.

The effect of over-expression of human LCAT on atherogenesis wassignificantly correlated with the changes in the plasma lipoproteins.The metabolism of the atherogenic apolipoprotein B (apoB) lipoproteinparticles is interrelated with that of the non-apoB associated highdensity lipoproteins. Patients with very high concentrations of eithertriglyceride-rich apoB particles (E. A. Brinton et al. (1991) J. Clin.Invest. 87:536) or with cholesterol-enriched LDL particles (J. R.Schaefer et al. (1992) Arteriosclerosis and Thrombosis 12:843) haveenhanced removal of HDL particles. The interrelationship of themetabolism of these particles is at least partially mediated by theesterification by LCAT of the cholesterol present in HDL and thesubsequent transfer of the cholesterol ester, by cholesterol estertransfer protein (CETP), from HDL to the apoB-containing particles. A.R. Tall (1993) J. Lipid Res. 34:125. Rabbits not only express more CETPthan rodents and humans, but they also increase the expression of thisprotein with cholesterol feeding. E.M. Quinet et al. (1990) J. Clin.Invest. 85:357. Thus, the over-expression of LCAT in these rabbits wasin the context of abundant CETP activity that affected the cholesterolconcentrations of both HDL and the non-HDL particles.

To further explore the relationships among the variables relevant tolipoproteins and atherogenesis, a series of bivariate Pearsoncorrelations were performed. The two atherosclerosis endpoints used inthis study were highly correlated for both the control rabbits as wellas for the entire study group (FIG. 4, pandls A and E). The severity ofatherosclerosis in both the control group (r=−0.64, p<0.006; FIG. 2,panel B) and the entire study group (r=−0.55, p=0.019; FIG. 4, panel F)was inversely related to the baseline LCAT activity. These inversecorrelations with LCAT activity were complemented by the significantdirect correlations of both the non-HDL (FIG. 4, panels C and G) and thetotal cholesterol/HDL cholesterol ratio (FIG. 4, panels D and H). Thesedose-response relationships strengthen the association betweenatherogenesis and the level of LCAT expression in the control as well asin the LCAT transgenic rabbits.

There are several causes for elevated concentrations of HDL cholesterol(hyperalphalipoproteinemia) and depressed concentrations of HDLcholesterol (hypoalphalipoproteinemia). The underlying etiology leadingto these different phenotypes may have different affects onatherogenesis. Although HDL cholesterol concentrations are principallydetermined by the clearance of HDL from the circulation (E. A. Brintonet al. (1994) Arterioscler. Thromb. 14:707; D. J. Radar et al., in HighDensity Lipoproteins: Physiopathology and Clinical Relevance, A. L.Catapano, F. Bemini and A. Corsini, Eds. (Raven Press, Ltd., New York,1993), p. 43), over-production of apoA-I can lead tohyperalphalipoproteinemia (D. J. Rader et al. (1993) Metabolism 42:1429)and protect against the development of atherosclerosis (E. M. Rubin etal. (1991) Nature 353:265). In addition, HDLs represent an array ofheterogeneous particles. In man, the HDL-containing apoA-I (LpA-I) hasbeen proposed to be more effective in reverse cholesterol transport thanparticles containing both apoA-I and apoA-II. R. Barbaras et al. (1988)Adv. Exp. Med. Biol. 243:271; J. C. Fruchart and G. Ailhaud (1992)Clinical Chemistry 38:793; H. B. Brewer, Jr. et al., in Disorders ofHDL, L. A. Carlson, Ed. (Smith-Gordon, 1990), p. 51. ApoA-I is a potentcofactor enhancing LCAT activity, and the modulation of LpA-I size issensitive to the presence of apoB-containing particles and LCATactivity. M. C. Cheung and A. C. Wolf (1989) J. Lipid Res. 30:499.Rabbits express no apoA-II (A. L. Borresen (1976) J. Immunogenet. 3:73;A. L. Borresen (1976) J. Immunogenet. 3:83; A. L. Borresen (1976) J.Immunogenet. 3:91), and these transgenic rabbits have only LpA-Iparticles. Over-expression of LCAT in these animals may have led to thegeneration of an anti-atherogenic HDL subspecies.

Transgenic Mice Expressing Human LCAT

The mouse, unlike the rabbit, is not an ideal model system for humanatherosclerosis. One reason for this is that lipoprotein metabolism inthe mouse is significantly different than that of rabbits, humans andother non-human primates. A key metabolic difference between rabbits andmice is the presence of cholesteryl ester transfer protein (“CETP”) inrabbits but not in mice. CETP mediates the transfer of cholesterylesters from HDL to apo-B containing lipoproteins in rabbits, but not inmice. CETP mediates the transfer of cholesteryl esters from HDL to apo-Bcontaining proteins, which facilitates delivery of cholesterol to theliver.

The effect of human LCAT expression on mice fed a high cholesterol dietwas tested. LCAT expression was effected by producing transgenic mice.Transgenic mice were made by microinjection of a 6.2 kb genomic fragmentof the entire human LCAT gene into fertilized eggs. See Vaisman et al.(1995) J. Biol. Chem. 270:12269.

Transgenic mice fed a diet high in cholesterol showed increased plasmatotal cholesterol, cholesteryl ester and apo-B-containing non-HDLlipoprotein concentrations, as did control animals. However, compared tocontrols, increased plasma total cholesterol and cholesteryl esterlevels in LCAT transgenic mice reflected primarily higher plasma HDLconcentrations, since the pro-atherogenic plasma non-HDL lipoproteinsdid not significantly differ between the two groups.

Despite relatively normal efflux of membrane cellular cholesterol, aswell as the persistence of higher HDL plasma concentrations in responseto the atherogenic diet, mice over-expressing human LCAT had enhancedatherosclerosis with increases in the mean aortic lesion size comparedto controls, and in contrast to transgenic rabbits, a model system forhuman atherosclerosis.

Increased HDL levels in humans are associated with a decreased risk ofcardiovascular disease. These results show that the mechanism by whichHDL is increased determines the anti-atherogenic properties of thelipoprotein. Therefore, increasing LCAT activity is likely to be mosteffective in mammals having similar lipoprotein metabolism as humans(e.g., rabbits and non-human primates) and in humans whose lipoproteinmetabolism (aside from LCAT deficiency) is relatively normal (e.g., nothaving CETP deficiency).

The present invention provides a novel method for use in theprophylactic or therapeutic treatment of atherosclerosis. While specificexamples have been provided, the above description is illustrative andnot restrictive. Many variations of the invention will become apparentto those skilled in the art upon review of this specification. The scopeof the invention should, therefore, be determined not with reference tothe above description, but instead should be determined with referenceto the appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

TABLE 1 M2 and M16 culture media for preimplantation embryos (FromLandel, supra.) I. Recipe for 100 ml each concentrated stock solutions.Make all solutions using sterile disposable plasticware anddouble-distilled or MilliQ water. Store at −20° C. Stock A 5.534 g NaCl0.356 g KCl 0.162 g KH₂PO₄ 0.293 g MgSO₄ · 7H₂O 2.610 g sodium lactate1.0 g glucose 0.060 g penicillin 0.050 g streptomycin Stock B 2.101 gphenol red 0.010 g NaHCO₃ Stock C 0.360 g sodium pyruvate Stock D 2.252g CaCl₂ · 2H₂O Stock E 5.958 g HEPES 0.010 g phenol red II. Preparationof 1X, M2 and M16 from concentrated stocks. Again, use sterile plasticware and very pure water. Mix components gently (do not shake whendissolving BSA, as it will foam), filter through a 0.45-μm filter, andstore 4° C. for up to 1 week. Stock M2 M16 A  1.0 ml 1.0 ml B 0.16 ml1.0 ml C 0.10 ml 0.10 ml  D 0.10 ml 0.10 ml  E 0.84 ml — H₂O  7.8 ml 7.8ml BSA    40 mg   40 mg

TABLE 2 CONTROL AND LCAT ACTIVITIES AND PLASMA LIPOPROTEINS BEFORE ANDAFTER CHOLESTEROL FEEDING Total High Non-High Cholesterol/ LCAT TotalTotal Density Density High Density ACTIVITY Cholesterol TriglycerideLipoprotein Lipoprotein Lipoprotein (nmol/ml/h) (mg/dl) Control (n = 9)Baseline 101 ± 11 29 ± 3  39 ± 4  24 ± 1  4 ± 3 1.17 ± 0.12Cholesterol-fed 98 ± 4 548 ± 57* 107 ± 15*  39 ± 3*  509 ± 57* 14.98 ±2.13* LCAT-Transgenic (n = 10) Baseline  1593 ± 101** 179 ± 7** 43 ± 4  161 ± 5** 18 ± 4 1.11 ± 0.02 Cholesterol-fed  308 ± 35* 396 ± 33* 81 ±8*  200 ± 21*  196 ± 14*  2.03 ± 0.07* From 5-7 ml of blood was drawn onrabbits after a 12 hour fast before (baseline) and after feeding a 0.3%cholesterol-chow diet (cholesterol-fed). α-LCAT activity was determinedusing 10 μl of plasma in a proteoliposome assay {{46885}}. EDTA plasmawas analyzed for total cholesterol and triglyceride concentrations(Sigma, St. Louis, MO) on a Hitachi 911 Autoanalyzer(Boehringer-Mannheim, Indianapolis, IN). The HDL cholesterolconcentration was determined on plasma that had been diluted withphosphate buffered saline 1:1 (v/v) and then precipitated with dextransulfate {{15590}}. The total plasma cholesterol concentration, and thenon-HDL cholesterol concentration was determined by subtracting the HDLcholesterol concentration from the total cholesterol concentration.*Differs from Baseline, p < 0.05; **Differs from Control Values, p <0.05

What is claimed is:
 1. A method for decreasing accumulation ofcholesterol in arteries in a human subject not suffering from alecithin:cholesterol acyltransferase (“LCAT”) deficiency syndromecomprising administering systemically to the subject a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and anamount of LCAT polypeptide effective to decrease the accumulation ofcholesterol in arteries of the subject.
 2. The method of claim 1 whereinthe pharmaceutical composition is administered parenterally.
 3. Themethod of claim 1 wherein the pharmaceutical composition is administeredby injection.
 4. The method of claim 1 wherein the pharmaceuticalcomposition is administered transmucosally or transdermally.
 5. Themethod of claim 1 wherein the pharmaceutical composition is delivered asa unit dosage form.
 6. The method of claim 5 wherein the unit dosage hasabout 10 mg to about 1000 mg of LCAT enzyme.
 7. The method of claim 6wherein the unit dosage form has about 40 mg to about 200 mg of LCATenzyme.
 8. The method of claim 1 wherein the LCAT is recombinant humanLCAT.